A central concern of the present post-genomic era of biology is understanding the chemical and physical mechanisms by which gene expression is regulated. Appropriate activation and repression of particular genes is necessary for maintaining normal cell function and is required for executing the programs of cell differentiation that are essential to the development of multicellular organisms. Collectively, gene regulatory systems are the "brain" of the cell that allow it to respond appropriately to environmental stimuli. Many cancers and other diseases result from deranged gene regulation. We here propose an entirely new approach to studying the molecular mechanisms of gene regulation in vitro. Instead of studying populations of molecules, we will directly visualize the regulatory machinery attached to an isolated single DNA molecule, following the progression of the machinery through its different states in real time while simultaneously observing the extent of transcriptional activation. Such direct visualization is made possible by novel multi-wavelength single-molecule fluorescence instrumentation newly developed our laboratory. This approach will allow us for the first time to elucidate regulation mechanisms by directly analyzing the dynamics of individual molecular interactions in complete regulatory complexes, instead of relying on inferences founded on data from piecemeal studies on individual proteins and their equilibrium interactions with DNA or with RNA polymerase. We will apply this technology to three different systems involved in regulation of transcription initiation and elongation in Escherichia coli. Each system was chosen because it is a prototype for a common mechanism of transcription regulation that functions analogously in both prokaryotes and eukaryotes. The proposed research will elucidate basic mechanisms of transcription regulation. In the long term this will improve public health by improving our understanding of human biology. In addition, the proposed research will help define the molecular basis for regulatory switches that affect virulence and environmental dissemination of human pathogens. This basic knowledge is expected to aid in the scientific research aimed at development of agents to combat infectious disease.